Psychiatric disorders, such as PTSD, depression and generalized anxiety disorder, are increasingly being recognized as dysfunctions of specific brain circuits, rather than alterations in global "brain chemistry." In order to develop new therapeutic approaches based on an understanding of underlying disease mechanisms, it is necessary to understand the normal function of the affected circuits. In this application, we propose to apply new, genetically based, techniques for manipulating neuronal function and mapping neuronal connectivity, to dissect the microcircuitry that underlies conditioned fear and its extinction. Our focus is on understanding the function of subpopulations of interneurons located in the central nucleus of the amygdala (CeA), a brain region involved in emotion. One subset of these neurons is marked by expression of protein kinase C-4 (PKC-4). Our preliminary data indicate that genetically based inactivation of these neurons enhances conditioned freezing, suggesting that these neurons may normally act to gate output from CeA. Using a recently developed genetic system for neuronal silencing, based on an ivermectin (IVM)-gated chloride channel, and an "intersectional" strategy to target expression of this heteromeric channel exclusively to PKC-4 cells in CeA, we will test this hypothesis and investigate the functional role of these neurons in fear learning and fear extinction, as well as in unconditional fear and anxiety (Specific Aim I). In Specific Aim II, we will further investigate the role of these neurons using neuronal activation strategies based on light (channelrhodopsin-2) or chemical activation. These experiments will test the necessity and sufficiency, respectively, of PKC-4 neurons in emotional behaviors mediated by the amygdala. In Specific Aim III, we will map the inputs and outputs to and from these neurons, using genetically based neuronal tracing and electrophysiological techniques. Finally, in Specific Aim IV we will test the hypothesis that activation of PKC-4 neurons is required for the behavioral effects of anxiolytic drugs, such as benzodiazepines. These studies should begin to provide a functional dissection of the amygdala at the level of granularity of specific neuronal subtypes, and may identify new cellular targets for therapeutic intervention in psychiatric disorders. PUBLIC HEALTH RELEVANCE: Psychiatric disorders, such as depression, schizophrenia and post-traumatic stress disorder (PTSD), exact a significant toll on public health, yet current methods to diagnose and treat them are inadequate. In order to develop a new generation of more effective treatments for these illnesses, with fewer side-effects, it is necessary to identify the underlying brain circuits that are impaired, understand the normal function of these circuits in emotional behavior, and describe how this function is altered in a given disorder. The present proposal applies an arsenal of new, genetically based, tools for dissecting neural circuit function at a level of specificity that has not previously been achieved, to understand the 'gating'mechanisms that control the flow of information through the amygdala, a brain structure important in learning (and "unlearning") fear.